Metagenomic insights into sulfate-reducing bacteria in a revegetated acidic mine wasteland

BackgroundThe widespread occurrence of sulfate-reducing microorganisms (SRMs, which are typically considered anaerobic organisms) in temporarily oxic/hypoxic aquatic environments indicates an intriguing possibility that SRMs can prevail in continuously oxic/hypoxic terrestrial environments rich in sulfate. However, little attention has been paid to such a possibility, leading to an incomplete understanding of microorganisms driving terrestrial part of the global sulphur cycle.ResultsIn this study, genome-centric metagenomics was employed to explore SRMs in a revegetated acidic mine wasteland under continuously oxic/hypoxic conditions. We reconstructed 12 Acidobacteria and four Deltaproteobacteria genomes encoding reductive DsrAB, of which five represented three new SRM genera. Our results showed that Acidobacteria-related SRMs differed considerably from Deltaproteobacteria-related SRMs in metabolic potentials. Genomes of Acidobacteria-related SRMs harbored more glycoside hydrolase (GH) genes than those of previously known SRMs. They also tended to encode more oxygen-tolerant hydrogenases and cytochrome c oxidases, but less methyl-accepting chemotaxis proteins (MCPs) than genomes of Deltaproteobacteria-related SRMs. More importantly, we discovered that SRM-infecting viruses can contribute to glycoside hydrolysis, chemotaxis and antioxidation of their hosts. Remarkably, one GH encoded by a SRM-infecting virus is responsible for the liberation of rhamnose (a monosaccharide that is accessible directly to SRMs for dissimilatory sulfate reduction) from plant cell-wall-derived oligosaccharides.ConclusionsTaken together, our results do not only improve our understanding of microorganisms driving dissimilatory sulfate reduction in terrestrial environments under continuously oxic/hypoxic conditions but also provides the first evidence for putative roles of viruses in S biogeochemical cycle in terrestrial ecosystems.

A Simple GC-MS/MS Method for Determination of Smoke Taint-Related Volatile Phenols in Grapes

Volatile phenols (VPs) derived from smoke-exposed grapes are known to confer a smoky flavor to wine. Current methods for determination of VPs in grape berries either involve complex sample purification/derivatization steps or employ two analytical platforms for free and bound VP fractions. We report here a simple gas chromatography-tandem mass spectrometry (GC-MS/MS) method for quantification of both free and bound VPs in grapes, based on optimized (1) GC-MS/MS parameters, (2) an analyte extraction procedure, and (3) phenol glycoside hydrolysis conditions. Requiring neither sample cleanup nor a derivatization step, this method is sensitive (LOD ≤ 1 ng/g berries) and reproducible (RSD < 12% for repeated analyses) and is expected to significantly reduce the sample turnover time for smoke taint detection in vineyards.

Computational simulation of mechanism and isotope effects on acetal heterolysis as a model for glycoside hydrolysis

DFT calculations for the equilibrium isotope effect for deuterium substitution at the anomeric centre Cα in 2-(p-nitrophenoxy)tetrahydropyran with continuum solvation show significant variation in the range of relative permittivity 2 ≤ ε ≤ 10. One-dimensional scans of potential energy (with implicit solvation by water) or of free energy (from QM/MM potentials of mean force with explicit aqueous solvation with a hybrid AM1/OPLS method) for heterolysis of the bond between Cα and the nucleofuge do not show a transition state. A two-dimensional free-energy surface that considers also the distance between Cα and a nucleophilic water indicates a pre-association DN*ANint‡ mechanism with a transition state involving nucleophilic attack upon an ion-pair intermediate, and this is supported by good agreement between the mean values of the calculated and experimental α-D KIEs. However, the magnitudes of the standard deviations about the mean values for the making and breaking C–O bonds suggest that the transition state is rather plastic, with Cα–Onu≈2 ± 0.4 Å and Cα–Olg≈3 ± 0.5 Å. Not only is nucleophilic solvent assistance necessary, but there is also evidence for electrophilic assistance through specific hydrogen bonding to the nucleofuge.

Methylglucosylation of aromatic amino and phenolic moieties of drug-like biosynthons by combinatorial biosynthesis

Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for “sugarcoating” various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase–methyltransferase (GT–MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana. The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both O- and N-glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4-O-methylglucosides. The resulting “unnatural products” show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell line-specific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats.

Identification of an isoflavone glycoside hydrolyzing b-glucosidase in the seeds of the desert legume Prosopis cineraria

Legumes, in general, are rich in glycosidic isoflavone conjugates, whereby specific b-glucosidases act to release the free aglycones that could serve in plant-microbe interactions and defense. Prosopis cineraria is an important legume tree, bearing medicinally useful and edible pods, generally growing in extreme arid/semi-arid zones of Rajasthan. However, specific phytochemical-cum-metabolic profiles are lacking for the same. Therefore, the present investigation undertook phytochemical and metabolic screening of the pods/seeds of P. cineraria for the presence of putative isoflavonoids viz. genistein and daidzein, their glycosides and b-glucosidase(s) capable of catalyzing the glycoside hydrolysis. Extraction and identification of these two aglycone forms of the above isoflavonoids were performed with solvent partition chromatography and Fluorescent/High Performance Thin Layer Chromatography, respectively. Furthermore, optimization of the isoflavone conjugate-specific b-glucosidase activity with respect to pH optima, temperature and time were carried out. The partially purified enzyme showed a temperature optima of 50°C and pH optima of 4.5. The enzyme also demonstrated activity towards natural substrates daidzin and genistin which are glycosides of isoflavonoids daidzein and genistein respectively. The methanolic extracts of the seeds of P. cineraria indicated the presence of related isoflavonoids which needs to be further validated.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.